Identification of the TRiC/CCT Substrate Binding Sites Uncovers the Function of Subunit Diversity in Eukaryotic Chaperonins

Spiess, Christoph; Miller, Erik J.; McClellan, Amie J.; Frydman, Judith

doi:10.1016/j.molcel.2006.09.003

Cited by 114 publications

(132 citation statements)

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“…TRiC from eukaryotes is a heterooligomeric, toroid shaped complex consisting of eight different subunits per ring and each subunit is ~ 50-60 kDa in size (Figure 6b). Interestingly, Group II chaperonins share sequence homology with GroEL at the ATP binding site, but they differ considerably in sequence of the substrate binding site (Kim et al, 1994;Spiess et al, 2006). TRiC, like GroEL, is an essential protein since at least two cytoskeletal proteins, actin and tubulin, are obligate substrates of TRiC (Dobrzynski et al, 1996;Gao et al, 1992;Llorca et al, 1999;Yaffe et al, 1992).…”

Section: Ii4113 the Chaperoninsmentioning

confidence: 99%

Firefly luciferase mutants as sensors of proteome stress

et al. 2011

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Section: Ii4113 the Chaperoninsmentioning

confidence: 99%

Firefly luciferase mutants as sensors of proteome stress

et al. 2011

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“…The largest divergence of sequence among the CCT subunits is in the apical substrate-recognition domain, suggesting that the different subunits of TRiC may have evolved distinctive subunit specificities (Frydman 2001;Kim et al 1994;Spiess et al 2006). Although only a few substrates have been studied, it is clear that not all CCT subunits are involved in binding of non-native-state substrates to TRiC (Feldman et al 2003;Hynes and Willison 2000;Llorca et al 2001;.…”

Section: Introductionmentioning

confidence: 99%

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee

Sergeeva

King

2013

Cell Stress and Chaperones

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Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1-8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing offpathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.

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“…Having eight distinct subunits may have allowed the diversification of substrate binding sites and activities within the ring of TRiC. Actin, tubulin (21,22), Huntingtin (8), and the von Hippel-Lindau tumor suppressor (23) are all recognized by distinct subsets of TRiC subunits through specific motifs; this directed binding may provide specificity for TRiC in the folding process. Accordingly, elucidating the subunit arrangement within the complex is essential to determine how TRiC affects the conformation and folding pathway of its substrates.…”

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4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Yao

Baker

Jakana

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ∼5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.asymmetric reconstruction | atomic model | subunit structure

show abstract

Identification of the TRiC/CCT Substrate Binding Sites Uncovers the Function of Subunit Diversity in Eukaryotic Chaperonins

Cited by 114 publications

References 38 publications

Firefly luciferase mutants as sensors of proteome stress

Firefly luciferase mutants as sensors of proteome stress

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

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